Introduction: The use of recombinant proteins in scientific research has increased greatly over the past few years. With this, the of techniques and products used for their amplification and purification have also grown. Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts (1). There are various factors that can affect the result of a protein purification experiment. This includes the lysis method, buffer composition, temperature, and protein solubility. To increase purity, usually a second binding/elution process is utilized; most frequently an ion-exchange (IEX) chromatography step is the method of choice. Depending on the pI-values and differences between target and contaminant proteins, anion-exchange or cation-exchange matrices are used in low salt concentrations to bind the target protein while contaminant proteins ideally bind weakly (2). The purpose of this lab was to overexpress a recombinant protein in bacteria. Competent bacterial cells were transformed with a DNA plasmid in order to grow a large culture of bacteria, which cells were harvested from. This protein was then purified using the affinity tag and Ni-NTA resin method. Gel electrophoresis was then used to analyze the samples. It was hypothesized that if the initiated bacterial cell population is transformed with a DNA plasmid, then a culture growth with the altered bacteria population will express the protein to a greater extent.
Materials & Methods: Ice bucket, 42oC water bath, gas burner, 2 x 14 ml clear, sterile round-bottom transformation tubes, 37oC incubator, colirollers, 2 LB Agar Amp plates, 1 spare Agar plate w/o antibiotic, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipette and pipette tips are needed for the first part. There will be 3 plates: one experimental plate with DNA and one Control plate that has no DNA to confirm that we have a clean technique and no colonies are growing without the plasmid. Place tubes of competent bacterial in ice bucket for 5 minutes. Briefly spin down the tube of plasmid DNA using a mini-centrifuge on the benchtop, then place it in ice and wait 30 minutes. Invert plates and place in 37 C incubator overnight. Set up burner to sterilize and add the appropriate amount of ampicillin to the two tubes of LB. Pick a single colony of bacteria growing and place them in your LB media. A bacterial culture will grow from this. The next day, you will harvest the cells and save them for the next lab. Lyse the E coli cells by adding a lysozyme to your cells. Distribute the lysate to several microcentrifuge tubes and centrifuge from 20 minutes at 14000 rpm at 4 C. Prepare a wash buffer and elution buffer. Then, purify the protein using a combination of batch and column chromatography. Using the provided reagents, strip the Ni-NTA. Using a nanodrop spectrophotometer, take a reading of your protein solution. You should have 6 samples collected. Centrifuge sample 1 for 5 minutes, and add the appropriate amount of loading buffer to samples 1-6. Using an electrophoresis, load your samples into the wells. After the gel electrophoresis is done, discard the buffers. Next day, dry the gel and take a picture of it.
Results:
Discussion:
The results of the first part of the experiment showed that the gbr22 DNA plasmid could be used to alter the E. coli bacteria such that the recombiant gbr22 protein could be expressed more. The use of heat-shocking the tube was important because it promoted the creation of pores in competent cell membranes. Through these pores, DNA would be able to get into the interior of the cells. The collirollers also were vital because they provided an even spread of the bacteria. Lysozyme was also added before storing the cells at a low temperature in order to ensure that the bacterial cell wall would remain traversable by the protein. Potential errors in this part of the experiment could possibly deal with time. The times that the solution was left in the ice and the time that the flask was stored in the freezer were longer than planned. Benzonase was added to digest the DNA and RNA, which reduced the viscosity of the mixture. The Ni-NTA mix was used as a binding agent for the HIS tags attached to the end of the negatively charged protein. This allowed for the creation of a larger molecule that would be prevented from flowing though the column. Due to the small amounts of imidazole added to both the wash and elution buffers, the majority of the gbr22 protein was never stripped from the Ni-NTA in the column, explaining why both the collected Elution 1 and Elution 2 liquids were virtually transparent. Additionally, the original elution buffer used was depleted throughout the course of the experiment, so a buffer produced later was used during the spectrophotometry procedure. This buffer, prepared with the correct amount of imidazole, contained more particulate matter than the original buffer, which is why the concentrations were perceived by the spectrophotometer to be negative – the elution buffer used to blank the machine contained more material than the actual elution. The data from the nanodrop results did, however, signify that absorbancy was higher at the maximal wavelength, which was to be expected. The purpose of the gel electrophoresis process was to characterize the purified proteins based on their respective molecular weights. A possible error that could of occurred that would impact the results of this part of the lab would be incorrect amounts of solution were added to the bacterial cells back in part 1. Another possible source of error could be not centrifuging the compound enough.
Conclusions:
The gbr22 protein was expressed in competent bacterial cells, purified and characterized in a gel to determine purity. In future experiments, protein expression and purification techniques can allow researchers to produce and analyze proteins. This could help Virtual Drug Screening because these proteins could possibly be used as drug targets or may serve other functions in other research.
The Life of the Purple Protein
Introduction:
The use of recombinant proteins in scientific research has increased greatly over the past few years. With this, the of techniques and products used for their amplification and purification have also grown. Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts (1). There are various factors that can affect the result of a protein purification experiment. This includes the lysis method, buffer composition, temperature, and protein solubility. To increase purity, usually a second binding/elution process is utilized; most frequently an ion-exchange (IEX) chromatography step is the method of choice. Depending on the pI-values and differences between target and contaminant proteins, anion-exchange or cation-exchange matrices are used in low salt concentrations to bind the target protein while contaminant proteins ideally bind weakly (2).
The purpose of this lab was to overexpress a recombinant protein in bacteria. Competent bacterial cells were transformed with a DNA plasmid in order to grow a large culture of bacteria, which cells were harvested from. This protein was then purified using the affinity tag and Ni-NTA resin method. Gel electrophoresis was then used to analyze the samples. It was hypothesized that if the initiated bacterial cell population is transformed with a DNA plasmid, then a culture growth with the altered bacteria population will express the protein to a greater extent.
Materials & Methods:
Ice bucket, 42oC water bath, gas burner, 2 x 14 ml clear, sterile round-bottom transformation tubes, 37oC incubator, colirollers, 2 LB Agar Amp plates, 1 spare Agar plate w/o antibiotic, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipette and pipette tips are needed for the first part. There will be 3 plates: one experimental plate with DNA and one Control plate that has no DNA to confirm that we have a clean technique and no colonies are growing without the plasmid. Place tubes of competent bacterial in ice bucket for 5 minutes. Briefly spin down the tube of plasmid DNA using a mini-centrifuge on the benchtop, then place it in ice and wait 30 minutes. Invert plates and place in 37 C incubator overnight. Set up burner to sterilize and add the appropriate amount of ampicillin to the two tubes of LB. Pick a single colony of bacteria growing and place them in your LB media. A bacterial culture will grow from this. The next day, you will harvest the cells and save them for the next lab. Lyse the E coli cells by adding a lysozyme to your cells. Distribute the lysate to several microcentrifuge tubes and centrifuge from 20 minutes at 14000 rpm at 4 C. Prepare a wash buffer and elution buffer. Then, purify the protein using a combination of batch and column chromatography. Using the provided reagents, strip the Ni-NTA. Using a nanodrop spectrophotometer, take a reading of your protein solution. You should have 6 samples collected. Centrifuge sample 1 for 5 minutes, and add the appropriate amount of loading buffer to samples 1-6. Using an electrophoresis, load your samples into the wells. After the gel electrophoresis is done, discard the buffers. Next day, dry the gel and take a picture of it.
Results:
Discussion:
The results of the first part of the experiment showed that the gbr22 DNA plasmid could be used to alter the E. coli bacteria such that the recombiant gbr22 protein could be expressed more. The use of heat-shocking the tube was important because it promoted the creation of pores in competent cell membranes. Through these pores, DNA would be able to get into the interior of the cells. The collirollers also were vital because they provided an even spread of the bacteria. Lysozyme was also added before storing the cells at a low temperature in order to ensure that the bacterial cell wall would remain traversable by the protein. Potential errors in this part of the experiment could possibly deal with time. The times that the solution was left in the ice and the time that the flask was stored in the freezer were longer than planned.
Benzonase was added to digest the DNA and RNA, which reduced the viscosity of the mixture. The Ni-NTA mix was used as a binding agent for the HIS tags attached to the end of the negatively charged protein. This allowed for the creation of a larger molecule that would be prevented from flowing though the column. Due to the small amounts of imidazole added to both the wash and elution buffers, the majority of the gbr22 protein was never stripped from the Ni-NTA in the column, explaining why both the collected Elution 1 and Elution 2 liquids were virtually transparent. Additionally, the original elution buffer used was depleted throughout the course of the experiment, so a buffer produced later was used during the spectrophotometry procedure. This buffer, prepared with the correct amount of imidazole, contained more particulate matter than the original buffer, which is why the concentrations were perceived by the spectrophotometer to be negative – the elution buffer used to blank the machine contained more material than the actual elution. The data from the nanodrop results did, however, signify that absorbancy was higher at the maximal wavelength, which was to be expected.
The purpose of the gel electrophoresis process was to characterize the purified proteins based on their respective molecular weights. A possible error that could of occurred that would impact the results of this part of the lab would be incorrect amounts of solution were added to the bacterial cells back in part 1. Another possible source of error could be not centrifuging the compound enough.
Conclusions:
The gbr22 protein was expressed in competent bacterial cells, purified and characterized in a gel to determine purity. In future experiments, protein expression and purification techniques can allow researchers to produce and analyze proteins. This could help Virtual Drug Screening because these proteins could possibly be used as drug targets or may serve other functions in other research.
References:
1. Rensselaer Polytechnic Institute Department of Biochemical Engineering. The Basics of Recombinant DNA. http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html (accessed Apr 17, 2013).
2. Protein production and purification.Nat Methods. 2008 Feb; 5(2):135-46.